When substrate inhibits and inhibitor activates: implications of β-glucosidases

β-glucosidases (BGs) catalyze the hydrolysis of β-glycosidic bonds in glucose derivatives. They constitute an important group of enzymes with biotechnological interest like supporting cellulases in degradation of lignocellulose to fermentable sugars. In the latter context, the glucose tolerant BGs are of particular interest. These BGs often show peculiar kinetics, including inhibitory effects of substrates and activating effects of inhibitors, such as glucose or xylose. The mechanisms behind the activating/inhibiting effects are poorly understood. The nonproductive binding of substrate is expected in cases where enzymes with multiple consecutive binding subsites are studied on substrates with a low degree of polymerization. The effects of inhibitors to BGs exerting nonproductive binding of substrate have not been discussed in the literature before.

Here, we performed analyses of different reaction schemes using the catalysis by retaining BGs as a model. We found that simple competition of inhibitor with nonproductive binding of substrate can account for the activation of enzyme by inhibitor without involving any allosteric effects. The transglycosylation to inhibitor was also able to explain the activating effect of inhibitor. For both mechanisms, the activation was caused by the increase of k_cat with increasing inhibitor concentration, while k_cat/K_m always decreased. Therefore, the activation by inhibitor was more pronounced at high substrate concentrations. The possible contribution of the two mechanisms in the activation by inhibitor was dependent on the rate-limiting step of glycosidic bond hydrolysis as well as on whether and which glucose-unit-binding subsites are interacting.

Knowledge on the mechanisms of the activating/inhibiting effects of inhibitors helps the rational engineering and selection of BGs for biotechnological applications. Provided that the catalysis is consistent with the reaction schemes addressed here and underlying assumptions, the mechanism of activation by inhibitor reported here is applicable for all enzymes exerting nonproductive binding of substrate.